Human alpha1-proteinase inhibitor (A1PI; also: alpha-1-antitrypsin) circulates at serum levels of 20 to 48 μM. The 51 kDa glycoprotein consists of 394 amino acids with three complex type N-glycans attached to asparagines 46, 83 and 247. Different N-glycan branching and truncation of the N-terminus result in a characteristic pattern on isoelectric focusing. In addition, a C-terminal truncated form of A1PI which misses the C-terminal lysine after cleavage by basic carboxypeptidases was described recently. A1PI is a member of the superfamily of serpins (serine proteinase inhibitors). Its anti-protease activity absolutely requires the active site Met358-Ser359. This site occupies an exposed loop on the molecule and tightly binds the active region of neutrophil elastase or other serine proteases including trypsin, cathepsin G, plasmin, thrombin and tissue kallikrein. A1PI exerts a preferential inactivating function for neutrophil elastase since the association rate constant for the A1PI/neutrophil elastase complex is 25-fold higher than those measured for other serine proteases. A1PI diffuses from plasma into the lung, where it is responsible for more than 90% of the anti-elastase protection of the lower respiratory tract. Deficiency of A1PI, leading to plasma concentrations below 20 μM, leaves the lung poorly protected and highly vulnerable to progressive lung destruction. Therapy is directed towards the replacement or augmentation of plasma A1PI.
The measurement of A1PI neutrophil elastase inhibitory activity was basically made possible as early as 1974 when the synthesis of the chromogenic substrate N-succinyl-L-alanyl-L-alanyl-L-alanin-p-nitroanilide (Suc(Ala)3-pNA) was described. This substrate has a high selectivity for elastase. Trypsin and chymotrypsin for example have hydrolysis rates of 0.007 and 0.014, respectively, when measured at the same concentration as elastase and setting the rate obtained with elastase to 100. At pH 8.0 and 25° C., the substrate Suc-(Ala)3-pNA has the kinetic constants Km and Kcat of 1.15 mM and 18.6 s−1 for elastase, respectively. Its selectivity and good solubility in water were the basis for an A1PI elastase inhibition assay as firstly described in 1981. This assay was based on the measurement of residual elastase activity of A1PI samples which have been incubated with an excess of elastase. Briefly, samples were incubated with a defined amount of porcine elastase for a defined time before the chromogenic substrate was added. The release of pNA was monitored photometrically and allowed to calculate the residual activity of elastase, which itself is inversely proportional to the A1PI activity of the sample. Standard assay formats using this chromogenic approach have a relatively high limit of quantification of about 3 μg/mL active A1PI. Although the sensitivity of such assay formats can be increased by using fluorogenic substrates for the measurement of residual elastase, they are still limited by the fact that one of the excess reaction partners, i.e. elastase, and not the reaction product is measured.
Therefore, a strong need exists to provide a novel assay for the determination of A1PI activity in a sample having improved sensitivity.
This need is satisfied by providing the embodiments characterized in the claims.